Chitosan is a linear polysaccharide composed of β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit), which primarily results from the alkaline deacetylation of chitin. Chitosan can exist in many structural conformations, depending on a variety of factors that include the degree of hydration, the electrolyte environment and the complexity of original chitin mixture. Chitosan and its amino-substituted derivatives are bioerodible, biocompatible and biodegradable cationic polymers that have been advanced for a wide variety of applications, including tissue engineering, drug and gene delivery, pharmaceutical formulation, scaffolds for cell growth and cell encapsulation, wound healing and surface hemostasis.
A well known property of chitosan is its solubility at acidic pH (<6) and insolubility at neutral pH, making its use in solution with living cells and tissues problematic. Various publications (Chenite, international patent application publication No. WO 99/07416; Chenite et al., 2000, Biomater., 21: 2155-2161; Chenite et al., 2001, Carbohyd. Polym., 46: 39-47) describe that admixing a polyol-phosphate dibasic salt, i.e. glycerol-phosphate (GP), to an aqueous solution of chitosan can increase the pH of the solution while avoiding precipitation of the polymer. In the presence of these particular salts, chitosan solutions of substantial concentration (0.5-3%) and high molecular weight (>several hundred kDa) remain liquid, at low or room temperature, for a long period of time with physiologically acceptable neutral pH region between 6.8 and 7.2. These chitosan-glycerol phosphate solutions which can gel upon mild heating (for example from 4 to 37° C.), are biocompatible, biodegradable and adhesive to human tissues, provide for new opportunities in the delivery of sensitive therapeutics.
Chitosan's properties allow it to rapidly clot blood, and have recently gained approval in the USA for use in bandages and other hemostatic agents. The first step in the early wound healing process is hemostasis due to blood coagulation. One approach to improve healing is to stabilize the blood clot by dispersing a soluble biocompatible polymer throughout uncoagulated fresh blood, the polymer acting as a scaffold throughout the clot to maintain its volume and to increase clot adhesion to tissues at the lesion site (Iliescu et al., 2007, Microsc. Res. Tech., 71: 236-247). Chitosan-GP solutions are non-toxic and bioresorbable, in addition to permitting coagulation and impeding clot retraction. They are mixed with autologous blood to form viscous chitosan-GP/blood mixtures that solidified in about 10 minutes in part due to the thrombogenicity of chitosan (Hoemann et al., 2007, Osteoarthritis & Cartilage, 15: 78-89).
Platelet-rich plasma (PRP) is a blood-derived product that concentrates a high number of platelets in a small volume of plasma. It has a substantial role in tissue regeneration and wound healing. In some applications, platelets have been used directly without being formulated. In other applications, a platelet gel (also known as a platelet clot) has been used that was prepared from PRP. The term “platelet gel” was introduced to describe the gelatin-like malleable product that results when thrombin and calcium are added to PRP. The addition of thrombin and calcium induces the cleaving fibrinogen to form fibrin which polymerizes, producing a glue-like gel. Platelets trapped in the gel are activated and release bioactive molecules. In most studies and clinical applications for tissue repair, a thrombin/calcium chloride solution was used to activate platelets to make PRP form a platelet gel. For example: PRP was clotted by adding a 10% thrombin solution (v/v, 1000 U/ml in 100 mM CaCl2) to yield a final thrombin concentration of 100 U/ml (Akeda et al., 2006, Osteoarthritis and Cartilage, 14: 1272-1280); 200 μl of PRP was treated with 20 μl of sterile bovine thrombin prepared by mixing 25 μl of the enzyme (1000 U/ml) in 10 ml of CaCl2 under sterile conditions to obtain a clot (Ranly et al., 2007, J. Bone Joint Surg. Am., 89: 139-147). PRP has also been activated just before application with 10% calcium chloride solution and 5000 units of bovine thrombin to form a gel (You et al., 2007, Oral Surg. Med. Oral Oral Pathol. Oral Radiol. Endo., 103: e8-e12).
In a few studies and clinical applications, only calcium was used to activate platelets to make PRP form a platelet gel (clot). For example: 10% CaCl2 solution was added into PRP to activate platelets and the platelet gel formed after 10 to 15 minutes (Casati et al., 2007, Int. J. Oral Maxillofac. Surg., 36: 132-136). Calcium chloride solutions with different concentration (5%, 10%, 25%, and 50%) have also been used alone to induce platelet activation, revealing that CaCl2 alone was suitable for triggering PRP activation, and that 5% calcium chloride solution was the most effective concentration in activating PRP to clot (Fedrico Luengo Gimeno et al., 2006, Thrombosis Journal, 4:18-25). PRP clots were also prepared by adding 0.5 ml of 10% calcium chloride to tubes and mixing with PRP. The mixture was then clotted with bone graft material graft (Human Freeze Dried Demineralized Cortical Bone) (Mendonca et al., 2006, Int. J. Oral Maxillofac. Surg., 35: 88-91). International patent application publication No. WO 96/23039 describes a novel type of hemostatic adhesive agents. These agents were prepared from PRP concentrate in combination with physiologically acceptable biocompatible polymers, which included alginates, poly-L-amino acids (poly-L lysine, poly-L-histidine and poly-α-D-glutamic acid), chitosan and chitin. In the presence of calcium ions, the combination of plasma concentrate with the polymers formed adhesives which had very strong adhesive properties and very rapid onset of these adhesive properties.
There is still a need for an improved polymer formulation with an improved solubility at physiological pH and ionic strength that can be mixed with PRP to form a hybrid polymer/PRP implant. There is a specific need for a hybrid polymer/PRP implant with good mechanical properties that can be obtained by a simple preparation method.